MYC induced nuclear antigen, isoform 2 (Mina53) has been shown to be a target gene of c-Myc, suggesting that it is involved in mamunalian cell proliferation (Tsuneoka et al. (2002) J. Biol. Chem. 277, 35450-9). Colon tumour cell lines were subsequently shown to highly express Mina53. Suppression of Mina53 expression severely suppressed proliferation of colon tumour cells in vitro and suppression of Mina53 or its activity may reduce cell proliferation and so be of use in the treatment of cancer. Elevated expression of Mina53 was therefore deemed a characteristic feature in colon cancer (Teye et al. (2004) Am. J. Pathol. 164, 205-16).
Reduction of Mina53 expression using RNAi has been shown to suppress cell proliferation in oesophageal squamous cell carcinoma (ESCC). Expression of Mina53 was also shown to be elevated in 83% of ESCC cell lines tested and patients with high expression level of Mina53 had shorter survival periods. Together, these results identify Mina53 as a potential prognostic factor for ESCC and indicate that inhibition of the unidentified role of Mina53 will be of benefit in treating cancer (Tsuneoka et al. (2004) Clin. Cancer Res. 10, 7347-56).
Mina53 was shown through immunoprecipitation to be part of a ribonucleoprotein complex where it associates with ribosomal as well as non-ribosomal proteins. It was proposed that Mina53 is involved in rRNA processing and/or ribosome assembly (Eilbract et al. (2005) Eur. J. Cell Biol. 84, 279-94). No molecular function or catalytic activity has been ascribed to Mina53.
None of the literature on Mina53 describes a method for expressing and purifying Mina53. The functional assays such as localization and immunoprecipitation carried out in the art do not require purified protein.
The 2-oxoglutarate (2-OG) and ferrous iron dependent oxygenases are a superfamily of enzymes that catalyse a wide range of reactions including hydroxylations, desaturations and oxidative ring closures (Hausinger (2004), Crit. Rev. Biochem. Mol. Biol. 39, 21-68; Ryle & Hausinger (2002) Curr. Opin. Chem. Biol. 6, 193-201; and Schofield et al. (1999) Journal of Inorganic Biochemistry 74, 49-49). Substrate oxidation is coupled to conversion of 2-OG to succinate and carbon dioxide. At least in some cases, binding of oxygen is followed by the oxidative decarboxylation of 2-OG to give succinate, CO2 and a ferryl species [Fe(IV)═O] at the iron centre. This highly reactive intermediate can then oxidize an unactivated C—H bond in the prime substrate, e.g. the oxidation of prolyl or asparaginyl residues in human proteins, or effect other oxidative reactions. Evidence for intermediates comes from substrate-analogue studies, model compounds and spectroscopic analyses.
The sequential binding of co-substrate and prime substrate, which is necessary to trigger oxygen binding, is probably important to limit the generation of reactive oxidizing species in the absence of prime substrate. The generation of such species in a prime-substrate-uncoupled manner can inactivate 2-oxoglutarate and the related oxygenases through self-oxidation, which sometimes leads to fragmentation. Typically, the uncoupled turnover of 2-OG occurs at approximately 5% of the rate of its coupled turnover in the presence of saturating concentrations of prime substrate, although it can also occur at a lower or higher rate.
Several 2-OG-dependent oxygenases, including procollagen prolyl hydroxylase, the hypoxia inducible factor prolyl hydroxylases, and anthocyanidin synthase, also have a requirement for ascorbate for full catalytic activity. Although ascorbate might stimulate activity by reducing Fe3+, or other high valent form of iron, to Fe2+ (either free in solution or at the active site), the stimulation of oxygenase activity by ascorbate might occur by other mechanisms, for instance, by promoting completion of uncoupled cycles. For uncoupled reaction cycles that are catalysed by procollagen prolyl hydroxylase in the absence of prime substrate, the oxidation of 2-OG to succinate has been shown to be stoichiometrically coupled to ascorbate. It is believed that one role of ascorbate is to function as a surrogate reducing substrate to ‘rescue’ the enzyme in the event of the uncoupled production of a ferryl [Fe(IV)═O] intermediate.
Studies with several enzymes have shown that certain substrate analogues and mutants can also stimulate uncoupled 2-OG turnover. It is also known in the literature that reducing agents other than ascorbate itself can act as reducing agents in the uncoupled turnover reaction, including derivatives of ascorbate (Zhang et al (1995) Biochem. J. 307 (Pt 1), 77-85 and Myllyla et al. (1978) Biochem. Biophys. Res. Commun. 83, 441-8).